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Inducible nitric oxide synthase (iNOS) in endotoxemia: chimeric mice reveal different cellular sources in various tissues¹

MICHAEL J. HICKEY², ELAINE SIHOTA^*, ABDELAZIZ AMRANI^*, PERE SANTAMARIA^*, LORI D. ZBYTNUIK^*, ELLA S. M. NG^*, WINNIE HO, KEITH A. SHARKEY and PAUL KUBES^*

Centre for Inflammatory Diseases, Monash University, Clayton, Australia; and the
^* Immunology and
Neuroscience Research Groups, University of Calgary, Calgary, AB, Canada

²Correspondence: Centre for Inflammatory Diseases, Monash University Department of Medicine, Monash Medical Centre, 246 Clayton Rd., Clayton, Vic., 3168, Australia. E-mail: michael.hickey{at}med.monash.edu.au

SPECIFIC AIMS

The primary aim of this study was to determine whether circulating leukocytes are a significant source of inducible nitric oxide synthase (iNOS) during the endotoxemic response. This issue was examined by creation of chimeric mice in which iNOS expression was restricted to either the leukocyte or parenchymal compartments.

PRINCIPAL FINDINGS

1. During the endotoxemic response, leukocytes make a significant contribution to total iNOS expression in the lung, but parenchymal cells are the dominant source of iNOS in peripheral organs (liver, colon, muscle)
Using bone marrow transfer between wild-type and iNOS^-/- mice, chimeric mice were generated in which iNOS expression was restricted to either the leukocyte compartment (leukocyte iNOS mice) or parenchymal cells (parenchyma iNOS mice). After allowing 8 wk for reconstitution of the bone marrow with donor cells (confirmed by flow cytometric analysis of thymocyte congenic markers), chimeric mice were treated with endotoxin (10 or 30 µg/kg, intravenous, 4 h) and tissues were removed for semiquantitative analysis of iNOS mRNA via RT-PCR and for immunohistochemical analysis of iNOS protein. iNOS mRNA was detectable in the blood of leukocyte iNOS mice, but not parenchyma iNOS mice, indicating that the lipopolysaccharide (LPS) treatment used was sufficient to induce iNOS expression by circulating leukocytes. Analysis of tissues removed from endotoxemic mice indicated that the relative contribution of leukocytes and parenchymal cells to total iNOS expression was tissue-specific. In the lung, leukocytes were responsible for at least 50% of the total iNOS mRNA expression observed (Fig. 1 ). Immunohistochemical analysis indicated that iNOS protein was detectable only in infiltrating leukocytes, not in resident parenchymal cells. However, in the peripheral organs examined [colon (Fig. 2 ), liver, and cremaster muscle], the major component of iNOS expression was derived from resident parenchymal cells.

View larger version (24K): [in this window] [in a new window]   Figure 1. Contribution of leukocyte- and parenchyma-derived iNOS to total tissue iNOS expression in the lung during endotoxemia. A) Representative gel of RT-PCR products from LPS-treated mice. iNOS and GAPDH products are shown in lung from WT intact, leukocyte iNOS, and iNOS-/- BMT mice. Parenchymal iNOS is not shown as it was run separately with appropriate controls. B) Relative units of iNOS expression in lungs of chimeric mice after 4 h treatment with 10 µg/kg LPS (top panel) and 30 µg/kg LPS (lower panel). Results are shown for WT intact mice representative of total iNOS expression; leukocyte iNOS mice, indicating leukocyte-derived iNOS; and parenchyma iNOS mice, indicating parenchymal iNOS. Data are shown as mean ± SE of 5–6 animals/group. ND denotes not detectable.

View larger version (24K): [in this window] [in a new window]   Figure 2. Contribution of leukocyte- and parenchyma-derived iNOS to total tissue iNOS expression in the colon during endotoxemia. A) Representative 2% agarose gel of RT-PCR products from LPS-treated mice. iNOS and GAPDH products are shown in colon from WT intact, leukocyte iNOS, and iNOS-/- BMT mice. B) Relative units of iNOS expression in colons of chimeric mice after 4 h treatment with 10 µg/kg LPS (top panel) and 30 µg/kg LPS (lower panel). Results are shown for WT intact mice, leukocyte iNOS mice, and parenchyma iNOS mice. Data are shown as mean ± SE of 5–6 animals/group. ND denotes not detectable.

2. Relative contribution of leukocyte-derived iNOS to total tissue iNOS expression is proportional to levels of leukocyte recruitment induced during endotoxemia
Chimeric mice were treated with endotoxin and tissues were removed for myeloperoxidase (MPO) analysis as an index of leukocyte recruitment. MPO levels in the lung were 20- to 30-fold higher than those observed in the colon and small intestine, indicating a much greater level of leukocyte recruitment in the lung. Similar results were found using immunohistochemistry to detect neutrophils. These data corresponded with the dominant contribution of leukocyte-derived iNOS to total iNOS expression in the lung. These findings suggest that the markedly elevated expression of leukocyte-derived iNOS in the lung may simply reflect the high number of leukocytes present in this tissue after endotoxin stimulation.

3. Systemic iNOS activity during endotoxemia, as indicated by plasma nitrite levels, is dominated by nonleukocytic cells
To address the issue of whether increased levels of iNOS in various tissues correlated with increased iNOS enzymatic activity at the systemic level, we measured plasma nitrite levels using HPLC. Nitrite is a breakdown product of nitric oxide synthase (NOS) activity, and its level in plasma increases significantly in endotoxemic mice. Experiments in iNOS^-/- mice have shown that this increase is predominantly due to iNOS activity rather than that of the constitutive NOS isoforms. After LPS treatment, plasma nitrite in parenchyma iNOS mice was not different from similarly treated wild-type mice. In contrast, nitrite levels in leukocyte iNOS mice, in which iNOS activity was limited to the leukocyte compartment, were consistently reduced by 40% from levels in wild-type mice. These findings indicated that parenchymal cells are the dominant source of endotoxemia-induced iNOS activity at the level of the systemic circulation.

CONCLUSIONS AND SIGNIFICANCE

The role of iNOS-derived nitric oxide in the sepsis response remains controversial. During sepsis, iNOS can produce high levels of NO and may play a role in sepsis-induced hypotension. There is some evidence that in the presence of the oxidant superoxide NO can react to form the potent cytotoxic oxidant, peroxynitrite. In contrast, it has been shown that iNOS can act as an endogenous protective molecule during a wide range of immune-mediated disease processes, including endotoxemia. One possible explanation for these conflicting observations is that different organs are affected differently by iNOS, potentially due in part to differing cellular sources and/or levels of induction of iNOS between different tissues. The latter is not trivial, as formation of peroxynitrite is thought to require coincident production of both NO and the superoxide anion. Leukocytes have the capacity to produce NO and superoxide, whereas other cells such as epithelial cells (intestinal and pulmonary), smooth muscle cells, and hepatocytes are less effective producers of superoxide. This unique ability of leukocytes could potentially make the contribution of leukocyte-derived iNOS a key determinant of the role of iNOS in the endotoxemic response.

Therefore, the aim of the present study was to analyze the separate contributions of iNOS expressed by either leukocytes or parenchymal cells to total iNOS expression during endotoxemia. A chimeric approach has allowed a clear distinction between these two sources of iNOS expression. In leukocyte iNOS mice, leukocytes were the only possible source of iNOS; in the parenchyma iNOS mice, only the parenchymal compartment (not circulating or infiltrating leukocytes) could express iNOS. Using this approach, we were able to demonstrate for the first time that during the early stages of the endotoxemic response, leukocyte-derived iNOS was only a minor contributor to total iNOS expression by most tissues except for the lung, despite the ability of circulating leukocytes to express iNOS in response to LPS stimulation. The expression data were supported by indirect analysis of systemic NOS activity (plasma nitrite analysis), which suggested that parenchymal cells were the dominant source of LPS-stimulated NO production at a systemic level. This indicates that despite the dominance of leukocyte-derived iNOS in total iNOS expression in the lung, this was not reflected in a comparable contribution of leukocyte-derived; iNOS to systemic levels of NOS activity.

It has been appreciated for some time that iNOS can be expressed by a wide range of cell types during various inflammatory responses. More recent observations have indicated that circulating leukocytes were also capable of iNOS expression during the sepsis/endotoxemia response. However, the relative contribution of iNOS expressed by circulating leukocytes to total iNOS expression has remained unknown. The data indicate that of the tissues we examined, the lung was unique in that a major component of the total iNOS expression was leukocyte derived. This observation is most likely accounted for by the dramatic increase in lung leukocyte content that occurs during endotoxemia. The lung is one of the primary sites of leukocyte trapping in endotoxemia. Indeed, the MPO analyses from the present study indicate that the relative amount of tissue leukocyte content in the lung is 20- to 30-fold higher than in the colon and small intestine. Therefore, the major contribution of leukocyte iNOS to total tissue iNOS in this tissue may simply reflect the relatively large number of leukocytes present in this tissue.

The above view does not negate the possibility that leukocytes recruited to the pulmonary microvasculature may respond to this microenvironment by up-regulating iNOS expression to a greater extent than in other vascular beds. In the lung, enormous surface areas are exposed to external stimuli, which may increase the level of iNOS expression in leukocytes present in the lung beyond that induced by the original endotoxin stimulus. This possibility is supported by the fact that many cell types require combined stimulation with two or more mediators for effective iNOS induction. The human lung is one of the few tissues in which continuous iNOS expression has been demonstrated in the absence of an obvious inflammatory response, indicating that the conditions in this tissue promote iNOS induction. Clearly the contention that the pulmonary microenvironment acts to enhance iNOS expression by leukocytes, either in the extravascular tissue or trapped within the microvasculature, cannot be discounted. Nevertheless, that we could detect iNOS in circulating cells suggests that a significant amount of iNOS was already being expressed by these cells before they were trapped in the lung.

In conclusion, this study provides a unique analysis of the contribution of the leukocyte and nonleukocyte compartments to endotoxemia-induced iNOS expression, clearly indicating that in tissues other than the lung, and at a systemic level, parenchymal cells are the dominant source of iNOS. Despite ongoing efforts, the role of iNOS-derived NO in endotoxemia-associated pathology remains poorly understood. Nevertheless, by more clearly defining the cellular sources of this molecule during the response, these experiments go some way toward furthering our understanding of the potentially organ-specific roles played by NO in endotoxemia.

View larger version (51K): [in this window] [in a new window]   Figure 3. Schematic diagram depicting the findings of this study. The upper portion depicts the different cell types able to express iNOS in response to endotoxemic stimulation. To determine the relative contributions of the leukocyte and parenchymal compartments to total iNOS expression and activity during the endotoxemic response, chimeric mice were created in which iNOS expression was restricted to either compartment. Subsequent examination of these mice during an endotoxemic response indicated that in peripheral organs (e.g., liver, colon), parenchymal cells were the dominant source of iNOS, whereas in the lung the majority of the iNOS detected was leukocyte derived. Finally, measurement of plasma levels of iNOS by-products indicated that parenchymal cells were the major source of iNOS activity at a systemic level.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0764fje; to cite this article, use FASEB J. (May 21, 2002) 10.1096/fj.01-0764fje.

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